Many different chemical substances are metabolized by the human body. Quite often, a number of chemical and/or biochemical reactions take place whereby a parent compound undergoes a plurality of structural changes, with one of the intermediate compounds thereof being a key factor in other but inter-related biochemical pathways. Accordingly, it becomes highly desirable to be able to determine, in a qualitative and/or quantitative fashion, the presence of various metabolic intermediates in a patient's biological fluid sample.
One commonly used approach to qualitatively detect and/or quantitatively measure the presence of a metabolic intermediate is through the use of a competitive protein binding assay. Usually, in order to carry out a competitive binding assay, the intermediate metabolite is obtained from a naturally-occurring source and/or synthesized from a precursor compound and is used in a labeled form for competitive binding to a complementary binder or receptor in competition with the unknown amount of unlabeled intermediate metabolite in the patient's sample. This necessitates an intermediate metabolite characterized by a sufficient degree of stability to be used as an assay reagent, that is, remaining substantially unchanged during a reasonable shelf life, say of at least one week, up to three to four weeks or longer.
As an example of an intermediate metabolite of the type discussed above, there may be mentioned N-5-methyltetrahydrofolic acid, an intermediate metabolite of folic acid. N-5-Methyltetrahydrofolic acid is a component of the blood and important to measure for diagnosing folic acid deficiency. Indeed, in man, folic acid and vitamin B.sub.12 are metabolically inter-related. It is essential to be able to determine the serum levels of N-5-methyltetrahydrofolic acid and vitamin B.sub.12 in order to indicate and treat megaloblastic anemia in man. Vitamin B.sub.12 and folate deficiencies are hematologically and clinically indistinguishable. Quite often, simultaneous assays are carried out for vitamin B.sub.12 and N-5-methyltetrahydrofolic acid.
The stability problem of N-5-methyltetrahydrofolic acid is exacerbated in the simultaneous vitamin B.sub.12 /folate assay because the preservative favored for stabilizing N-5-methyltetrahydrofolic acid, ascorbic acid, interferes with competitive binding vitamin B.sub.12 analysis.
The prior art discloses sealing sensitive intermediate metabolites such as N-5-methyltetrahydrofolic acid in lyophilized form in ampoules under a nitrogen atmosphere. Such a technique is very impractical in preparing a reagent kit, plus it would not prolong stability after reconstitution, which for N-5-methyltetrahydrofolic acid is only about three days at 4.degree. C.